Human Immunodeficiency Virus (HIV) Separation and Enrichment via the Combination of Antiviral Lectin Recognition and a Thermoresponsive Reagent System

• Published in: Pharmaceutical research 2016-10, Vol.33 (10), p.2411-2420
• Main Authors: Phan, Joseph C., Nehilla, Barrett J., Srinivasan, Selvi, Coombs, Robert W., Woodrow, Kim A., Lai, James J.
• Format: Article
• Language: English
• Subjects: Antiviral agents; Antiviral Agents - metabolism; Bacterial Proteins - metabolism; Bioassays; Biochemistry; Biomedical and Life Sciences; Biomedical Engineering and Bioengineering; Biomedicine; Carrier Proteins - metabolism; Chemical tests and reagents; Diagnosis; Ethylenediaminetetraacetic acid; HIV; HIV (Viruses); HIV testing; HIV-1 - isolation & purification; HIV-1 - metabolism; Human immunodeficiency virus; Humans; Ionization; Lectins; Lectins - metabolism; Lentivirus; Matrix; Medical Law; Nanoparticles; Pharmacology/Toxicology; Pharmacy; Protein Binding - physiology; Proteins; Research Paper; Surface Plasmon Resonance - methods; Temperature
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1007/s11095-016-1980-7

Purpose In order to improve the detection limit of existing HIV diagnostic assays, we explored the use of a temperature-responsive magnetic nanoparticle reagent system in conjunction with cyanovirin-N for HIV recognition to rapidly and efficiently concentrate viral particles from larger sample volumes, ~ 1 ml. Methods Cyanovirin-N (CVN) mutant, Q62C, was expressed, biotinylated, and then complexed with a thermally responsive polymer-streptavidin conjugate. Confirmation of protein expression/activity was performed using matrix assisted laser desorption/ionization (MALDI) and a TZM-bl HIV inhibition assay. Biotinylated CVN mutant recognition with gp120 was characterized using surface plasmon resonance (SPR). Virus separation and enrichment using a thermoresponsive magnetic nanoparticle reagent system were measured using RT-PCR. Results Biotinylated Q62C exhibited a K D of 0.6 nM to gp120. The temperature-responsive binary reagent system achieved a maximum viral capture of nearly 100% HIV, 1 × 10 5 virus copies in 100 μl, using pNIPAAm-Q62C within 30 minutes. Additionally, the same reagent system achieved nearly 9-fold enrichment by processing a 10-times larger sample of 1000 μl (Fig.  3 ). Conclusion This work demonstrated a temperature-responsive reagent system that provides enrichment of HIV using antiviral lectin CVN for recognition, which is potentially amenable for use in point-of-care settings.